Yong Liu

Background: HBeAg‐positive chronic hepatitis B (CHB) with low HBsAg levels represents a relatively rare serological pattern and is closely associated with the severity of liver disease. However, the underlying mechanisms in such cases remain largely unclear. Methods: Treatment‐naïve HBeAg‐positive CHB patients with low HBsAg levels in China were enrolled and analysed. In vitro cell experiments and immunoassays were conducted to investigate the effects of the preS2 deletion mutation on virus reproduction and host immune response. Results: Treatment‐naïve HBeAg‐positive CHB patients with low HBsAg levels (low HBsAg group) exhibited higher fibrosis scores and a greater prevalence of quasispecies mutations introduced by preS2 deletion compared to patients with positive HBeAg and high HBsAg levels (high HBsAg group). Further analysis revealed that fibrosis scores in CHB patients with the preS2 deletion mutations were significantly higher compared to both in wild‐type patients and the high HBsAg group. In vitro assays indicated that while this mutation may not impact HBV replication, it significantly reduced viral infectivity. The number of viral‐specific IFN‐γ‐secreting cells induced by the mutant was significantly lower than that induced by the wild‐type strain. Additionally, the levels of HBs‐specific B cells and cytokine secretion from lymphocytes triggered by the mutant strain were significantly reduced. Conclusions: HBeAg‐positive CHB patients with low HBsAg and genotype C exhibited higher noninvasive fibrosis indexes compared with typical patients, accompanied by a significant increase in quasispecies variants associated with preS2 deletion. The emergence of the preS2 deletion mutants in patients could be due to its enhanced ability to evade the host immunity.

Here, we regularly followed two SARS-CoV-2 infected cohorts to investigate the combined effects of neutralizing antibodies (NAbs) and B and T cell profiles during the convalescent period. Ten infected participants in December 2022 were selected to assess the effects of an inhaled adenovirus type 5 vectored COVID-19 vaccine (Ad5-nCoV) booster on B cells and humoral immunity in the first cohort. To evaluate T cell responses, eight primary and 20 reinfection participants were included in the second cohort. Blood samples from all 38 participants were collected at 1-, 2-, and 6-months post-infection. In the first cohort, eighteen monoclonal antibodies (mAbs) with neutralizing activity from memory B cells (MBC) against SARS-CoV-2 mutants were obtained by high throughput single-B-cell cloning method, which lasted from 1- month to 6- month post infection. The overall number of mAbs from MBC in the boosted immunization group was higher than that in the nonboosted immunization group at 2-, and 6-months post-infection. In the second cohort, circulating T follicular helper cells (cTfh) and AIM ⁺CD4 ⁺T cells increased over time in the reinfection group (P < 0.05). In both cohorts, serum NAb titers showed significant immune escape, while cTfh and AIM ⁺CD4 ⁺T cells in the second cohort essentially showed no immune escape to new strains (including XBB, EG.5). AIM ⁺CD4 ⁺T cells against BA.5 and EG.5 were strongly negatively correlated with the time to viral clearance in the reinfected group at 6-months post-infection. We comprehensively assessed the ability of the SARS-CoV-2 boosted immunization and reinfection-induced generation of T/B cell immune memories in preventing reinfection.

Background/Aims: C-C motif ligand 5 (CCL5) is reported to play a key role in acute and chronic liver diseases. However, the association between the CCL5 and chronic hepatitis B (CHB) remains to be explored. We aimed to investigate the CCL5 expression in the liver tissues of CHB patients and compared the CCL5 expression among CHB patients with different stages of liver inflammation and fibrosis. Materials and Methods: Liver tissue specimens from fifty-one CHB patients who underwent liver biopsy and twelve healthy liver donors were included in the present study. CCL5 expression in the liver tissues was analyzed using immunohistochemistry. The hepatic inflammation grades and fibrotic stages of CHB patients was assessed by the Scheuer classification system. Results: Livers of CHB patients exhibited a significantly accumulated CCL5+ cells when compared to that of healthy controls (42.80±4.37 vs 7.25±0.99/HPF, p<0.001). CHB patients with higher hepatic inflammation grades had more CCL5+ cells in their livers compared to those with lower grades (p<0.05). However, the numbers of CCL5+ cells were not correlated with the fibrotic stages in CHB patients (r=0.073, p=0.61). The number of CCL5+ cells in the liver tissues of CHB patients were positively correlated with alanine transaminase levels (r=0.278, p=0.041) and aspartate aminotransferase levels (r=0.328, p= 0.009). Conclusion: CHB patients have a significant accumulation of CCL5+ cells in the liver, and CCL5 may play a pathological role in hepatic inflammation of CHB.

B cell hyperactivation and functional impairment were identified from patients with chronic hepatitis B virus (CHB) infection; however, the underlying mechanism remains unknown. Here, we aim to elucidate the mechanisms responsible for B cell hyperactivation during HBV infection. Peripheral CD19⁺ B cells isolated from 4 CHB patients and 4 healthy volunteers were analysed by RNA sequencing. A total of 1401 differentially expressed genes were identified from B cell transcriptome of CHB patients vs healthy volunteers. We found that B cells from CHB patients were functional impaired, with increased TLR4 expression, activated NF‐κB pathway and altered mitochondrial function. The expression of B cell activation‐related genes, including TLR4, was further validated using additional clinical samples. To further verify the role of TLR4 in B cell activation during CHB, B cell phenotypes were determined in wild‐type (WT) and TLR4^−/− HBV‐carrier mice. Hyperactivated B cell and TLR4 signalling pathway were observed in WT HBV‐carrier mice, while TLR4 ablation failed to induce B cell hyperactivation, and downstream MyD88 and NF‐κB were also not altered. Taken together, TLR4 pathway plays a pivotal role in B cell hyperactivation during CHB, which might serve as a promising target for B cell function restoration.

Disruptions of angiogenesis can have a significant effect on the healing of uterine scars. Human endometrial perivascular cells (CD146+PDGFRβ+) function as stem cells in the endometrium. Cysteine-rich angiogenic inducer 61 (CYR61) plays an important role in vascular development. The purpose of this study was to observe the effects of the transplantation of human endometrial perivascular cells (En-PSCs) overexpressing CYR61 on structural and functional regeneration in rat models of partial full-thickness uterine excision. We first sorted human En-PSCs from endometrial single-cell suspensions by flow cytometry. Human En-PSCs expressing low or high levels of CYR61 were then generated via transfection with a CYR61-specific small interfering ribonucleic acid (si-CYR61) construct or overexpression plasmid. To establish a rat model of uterine injury, a subset of uterine wall was then resected from each uterine horn in experimental animals. Female rats were randomly assigned to five groups, including a sham-operated group and four repair groups that received either PBS loaded on a collagen scaffold (collagen/PBS), En-PSCs loaded on a collagen scaffold (collagen/En-PSCs), En-PSCs with low CYR61 expression loaded on a collagen scaffold (collagen/si-CYR61 En-PSCs), and En-PSCs overexpressing CYR61 loaded on a collagen scaffold (collagen/ov-CYR61 En-PSCs). These indicated constructs were sutured in the injured uterine area to replace the excised segment. On days 30 and 90 after transplantation, a subset of rats in each group was sacrificed, and uterine tissue was recovered and serially sectioned. Hematoxylin and eosin staining and immunohistochemical staining were then performed. Finally, the remaining rats of each group were mated with fertile male rats on day 90 for a 2-week period. Sorted En-PSCs expressed all recognized markers of mesenchymal stem cells (MSCs), including CD10, CD13, CD44, CD73, CD90, and CD105, and exhibited differentiation potential toward adipocytes, osteoblasts, and neuron-like cells. Compared with En-PSCs and En-PSCs with low CYR61 expression, En-PSCs with elevated CYR61 expression enhanced angiogenesis by in vitro co-culture assays. At day 90 after transplantation, blood vessel density in the collagen/ov-CYR61 En-PSCs group (11.667 ± 1.287) was greater than that in the collagen/En-PSCs group (7.167 ± 0.672) (P < 0.05) and the collagen/si-CYR61 En-PSCs group (3.750 ± 0.906) (P < 0.0001). Pregnancy rates differed among groups, from 40% in the collagen/PBS group to 80% in the collagen/En-PSCs group, 12.5% in the collagen/si-CYR61 En-PSCs group, and 80% in the collagen/ov-CYR61 En-PSCs group. In addition, four embryos were evident in the injured uterine horns of the collagen/ov-CYR61 En-PSCs group, while no embryos were identified in the injured uterine horns of the collagen/PBS group. The results showed that CYR61 plays an important role in angiogenesis. Collagen/ov-CYR61 En-PSCs promoted endometrial and myometrial regeneration and induced neovascular regeneration in injured rat uteri. The pregnancy rate of rats treated with transplantation of collagen/En-PSCs or collagen/ov-CYR61 En-PSCs was improved. Moreover, the number of embryos implantation on the injured area in uterus was increased after transplantation of collagen/ov-CYR61 En-PSCs.

During the natural history of chronic hepatitis B infection (CHB), the function of B cells is still obscure. Several limited studies have suggested that B cells are highly active in patients with CHB. In the present study, we reported that the 4‐1BB ligand (4‐1BBL) expression on B cells was significantly higher in patients with CHB than that in healthy subjects, meanwhile, the patients with CHB had higher serological IgG levels. Further, after being stimulated with sCD40L or hepatitis B core antigen (HBcAg), B cells had higher levels of 4‐1BBL. After being cocultured with 4‐1BBL+ B cells, the expressions of CD69 and 4‐1BB on CD4+ T cells were significantly higher than that cocultured with 4‐1BB− B cells. Cytokines including interleukin (IL)‐2, IL‐4, and IL‐6 were significantly higher in the supernatant from 4‐1BBL+ B cells coculture group than those from coculture group of 4‐1BBL− B cell group, respectively; while IFN‐γ and TNF‐α in cocultured supernatant of 4‐1BBL+ B cell group were significantly lower. Consistently, the total IgG levels in culture supernatant were significantly higher in 4‐1BBL+ B cell group. Thus, hyperactive status of B cells in patients with CHB could be partially derived from the higher 4‐1BBL expression on B cells triggered by HBcAg. 4‐1BBL signaling pathway is involved in B cells activation, and further regulates B cell‐T cell interaction by modulating the cytokines secretion, which might be critical in B cells dysfunction during CHB infection.

To observe longitudinally the expression of Programmed death 1(PD-1) on peripheral blood T cells in chronic hepatitis B patients underwent antiviral treatment with entecavir (ETV) and to explore the relationship between PD-1 expression and HBeAg seroconversion. Twenty HBeAg positive patients underwent antiviral treatment with ETV were followed up for 51 weeks. 14 patients remained HBeAg positive and 6 patients achieved HBeAg seroconversion. Peripheral blood was collected at six time points: T0: baseline, T1: 2-4week; T2: 5-10week; T3: 11-20week; T4: 21-30week: T5: 31-51week. PD-1 expressions on T cells were assessed by flow cytometry. Serum HBV DNA loads were determined by real-time fluorescent quantitative polymerase chain reaction (PCR) and serum ALT levels were examined at the same time. At baseline, serum HBV DNA load of patients without HBeAg seroconversion and with HBeAg seroconversion were (7.54+/-0.67) log10 copies/ml and (7.30+/-0.79) log10 copies/ml (P more than 0.05), the ALT levels were (187.26+/-184.15) U/L and (272.17+/-215.07) U/L (P more than 0.05), PD-1 exprissions on CD4+ T cells were 6.04%+/-3.71% and 6.77%+/-2.88% (P more than 0.05), PD-1 exprissions on CD8+ T cells were 6.39%+/-3.33% and 8.88%+/-2.84% (P more than 0.05). After ETV treatment, serum HBV DNA loads and ALT levels both decreased gradually, which was positively correlated with PD-1 expressions on CD4+ and CD8+ T cells (r=0.212, P = 0.05; r=0.377, P less than 0.01; r=0.279, P less than 0.05; r=0.347, P less than 0.01). During the same monitoring period, the HBV DNA loads, ALT levels and PD-1 expressions on T cells of the patients with HBeAg seroconversion decreased significantly as compared with the patients without HBeAg seroconversion. Besides, the decrease of HBV DNA loads during period deltaT0-T1 and deltaT0-T2 and PD-1 expressions on CD8+ T cells during period deltaT0-T2 and deltaT0-T3 were significantly different between these two kinds of patients (49.9% vs 37.3%, P less than 0.05; 56.7% vs 47.4%, P less than 0.05; 70.1% vs -4.2%, P less than 0.05; 66.9% vs 24.5%, P less than 0.05). The rapid decrease of PD-1 expression on peripheral CD8+ T cells after antiviral treatment with ETV is positvely correlated with the decrease of serum HBV DNA loads and may be used as a predictive index for HBeAg seroconversion in HBeAg positive patients.